The long term objective of this project is to identify the molecular defect(s) within the spectrin membrane skeleton of irreversibly sickled cells which cause these cells to lock into the sickled shape. In preliminary studies we have demonstrated that core skeletons which contain only spectrin, protein 4.1, and actin remain sickled when isolated from high density sickled cells. Furthermore, we have demonstrated that spectrin membrane skeletons. from normal red cells dissociate when prepared at 37 degrees C, while the skeletons from high density sickle cells remain associated- The fact that irreversibly sickled core skeletons remain associated at 37 degrees C is not due to covalent linkages; because these skeletons dissociate completely to spectrin, protein 4.1, and actin in the presence of SDS. Our hypothesis is that posttranslational modification(s) of spectrin, protein 4.1, or actin lead to an irreversibly sickled skeleton which resists dissociation at 37 degrees C. We will test this hypothesis with the following studies. The morphology of normal and sickle cell core skeletons prepared at 0 degrees C and 37 degrees C will he studied by negative staining and electronmicroscopy. The protein which is functionally defective will he identified by [1] studying the spectrin-4.1-f-actin interaction and spectrin dimer-dimer interaction when spectrin is isolated from control versus sickled cells. [2] Studying the spectrin-4.1-f-actin interaction when protein 4.1 is isolated from control versus sickled cells. [3] Determining the rate of polymerization of sickle cell versus control actin, and the ability of sickle cell versus normal actin protofilaments to serve as seeds for actin polymerization. Once the abnormal protein(s) have been identified the posttranslational modification(s) causing the problem will he identified by reverse phase HPLC separation of proteolytic digests of the sickle cell versus control skeletal protein, followed by protein sequencing of altered peptides by MS/MS mass spectrometry.